180 DB. J. GILCHBIST 01^ THE MINUTE STBFCTUBE OF 



they were overcome ; but sliall merely describe the metliod whicli 

 I ultimately found workable. 



Au important factor seems to be the kind of blue used, as, 

 out of the several sorts tried, one (the Koclist) gave much better 

 results than the others. 



A nearly saturated solution of the blue was made in distilled 

 water (not salt solution), and about 2 cub. cent, of it injected 

 into the living animal. It was found that this cau be done best 

 through the muscular foot, as the puncture is soon closed and 

 escape of the injecting material prevented. The animal thus- 

 injected was put back into sea-water and left for an hour or 

 two ; and it was found advisable to operate on several animals at 

 once, if material were abundant. The nervous system could then 

 be laid bare, or dissected out and laid on a slide, just enough 

 water being added to keep the preparation from drying up. 

 At times no coloration was at first observed, but soon (by oxi- 

 dation) the blue appeared. This generally happened in a few 

 minutes ; and the process could be easily watched under the 

 microscope and stopped at any stage. It was found that, as a 

 rule, the outside of the ganglia became coloured only before or 

 when the colour had spread from the nerve into the ganglion- 

 cells. This outside bad therefore to be removed, as fortu- 

 nately was possible by embedding and cutting with a microtome 

 in the usual way, thanks to Dr. Bethe's method of fixing the 

 stain with ammonium molvbdate ; but there was thus introduced 

 another possible difficulty, in addition to that caused by the erratic 

 selection of the elements by the blue, as fibres or cell-processes 

 once cut were very difficult to determine in the next section. 

 Both difficulties, bowever, w^ere overcome by making abundant 

 preparations, and by cutting thick sections ('5 millim.) which 

 to a certainty would include complete cells. The cut parts, 

 moreover, could be readily recognized by their abrupt endings. 



The central nervous system having been thus got at, it re- 

 mained to find a method for demonstrating the peripheral nervous 

 system, and the sensory cells of the epidermis and the higher 

 sense-organs. Since the essential point was to get the blue in 

 direct contact with the elements to be stained, it appeared from 

 the afore-mentioned trials that this might best be assured by 

 injecting through the blood-system. Accordingly a large quantity 

 of the solution was forcibly injected until the blue showed through 

 the epidermis. A piece of the tissue thus coloured, treated as 



